The present invention is directed to apparatus and methods for sample delivery. More specifically, the invention is directed apparatus and methods for automated microscale sample delivery to chemical reagents and/or analytical apparatus.
Methods for conducting chemical reactions often require multiple steps in multiple reaction vessels involving extensive handling of reagents. These limitations may result in experimental error, contamination, and a risk of exposure of laboratory workers to hazardous substances.
Analytical techniques typically require a high degree of labor and the use of complex apparatus. Moreover, many laboratory and industrial chemical processes involve the use of relatively large volumes of reagents and multiple laboratory instruments. Typical large scale immunoassays, e.g., require the use of pipettes, reagent vessels, and reaction chambers. See, e.g., Mattiasson et al., Proc. Int. Symp. on Enzyme-Labeled Immunoassay of Hormones and Drugs, (Pal, S., Ed., Walter de Gruyter, Berlin (1978), p. 91). Such processes, regardless of the size of the reaction, also may require multiple steps. Accordingly, there is a potential for reduced accuracy due to the introduction of impurities, volumetric inaccuracies, and low reproducibility. These problems especially are acute in microscale diagnostic applications in which biological samples are analyzed, such as, e.g., immunoassays, polynucleotide amplifications, or hybridizations.
Recently, efforts have been made to streamline chemical processes to reduce costs, increase accuracy, and improve reaction yields. For example, capillary electrophoresis techniques have been proposed to increase resolution in immunoassays. Various attempts have been made to enhance other common analytical techniques, such as the polymerase chain reaction (PCR). For example, U.S. Pat. No. 5,273,907 reports a capillary pre-loaded with PCR reagents which is used to deliver a sample to the reagents for DNA amplification. Similarly, International Patent Publication WO 93/22058 describes a microscale device for performing PCR. In this case, PCR reagents from a first chamber are mixed with sample in a second chamber by movement of materials through channels in a microchip.
There is a need in the art for methods and apparatus which decrease the labor, cost, biohazard exposure and complexity associated with microscale sample delivery for chemical reactions and analytical techniques. The present invention addresses these needs.
Apparatus and methods now have been developed to deliver automatically a sample to a reaction vessel, an analytical device or any location where sample introduction or deposition is desired. Broadly, the invention relates to a thermally-controlled microscale sample delivery system and methods of its use. An embodiment of the invention includes apparatus and methods for delivery of a sample to a channel wherein chemical reactions occur.
A sample delivery system of the invention generally comprises a housing defining a channel, e.g., a capillary, and a temperature control device in thermal communication with the channel. The channel preferably is closed at one end, and contains an opening for introduction of a sample. The closed end typically is associated with the temperature-control device. The temperature control device may be a thermoelectric heater, such as a Peltier element, for heating and cooling a thermally expandable fluid in the channel. The temperature control device also may include a temperature controlled fluid which is in thermal communication the channel.
A sample delivery system of the invention preferably includes an array of independent channels so multiple samples simultaneously may be delivered. The channels often are capillaries which may have non-wettable surfaces. In another embodiment of the invention, the channels are capillaries having immobilized therein at least one chemical reagent. In a preferred embodiment, a reagent is pre-loaded into a capillary by immobilization e.g., by drying the reagent on the walls of the capillary. Reagents also may be immobilized by absorption into a plug of material, such as cotton, which is placed in the capillary. Reagents typically are immobilized on the capillary walls in one or more discrete locations.
Another embodiment of the sample delivery system includes a second temperature control device. The second temperature control device may be positioned for heating and/or cooling the sample and reagents in the capillary. The second temperature control device preferably comprises a first conduit for heating and a second conduit for cooling. The second temperature control device, therefore, is adapted for controlling the temperature in a discrete portion of the capillary, typically towards the open end of the capillary. That is, the second temperature control device should not induce temperature changes of the fluid or gas near the closed end of the capillary. To this end, a sample delivery system also may include an insulator partition within the capillary to assist in maintaining a volume of sample stationary within the capillary.
Accordingly, the local temperature of a reaction between reagents and sample may be controlled by a second temperature control device without moving the sample in the capillary or by moving the second temperature control device along the capillary. A sample delivery system of the invention may contain more than two temperature control devices. However, a single temperature control device may be used to heat and/or cool both the entire capillary and discrete locations. A sample delivery system of the invention may contain more than two temperature control devices.
Methods of the invention provide for the delivery of a sample to predisposed reagents within a channel, to an input port of an analytical device or to another location where the sample or its reaction products are desired. In a preferred embodiment, a temperature control device in association with a capillary heats the gas in the capillary so that the volume occupied by the gas increases, thereby increasing its pressure. This increase in volume and pressure forces gas through the capillary opening. The opening of the capillary then is exposed to a sample, e.g., by submerging the open end in a liquid sample. Upon cooling, the volume of gas remaining in the channel contracts, and the pressure within the capillary decreases. Consequently, an aliquot of sample is drawn into the capillary to fill the volumetric void left by the contracting gas. If sufficiently cooled, the sample is drawn far enough into the capillary to contact chemical reagents disposed therein, if present.
Products of the reaction, if present, can be removed from the capillary by heating the gas near the closed end of in the capillary. Alternatively, the sample drawn into the capillary may be removed from the capillary without a reaction occurring and be delivered to another reaction site, capillary, analytical device or anywhere sample deposition is desired.
In another preferred embodiment, a sample delivery system of the invention is used to introduce a sample into a sample analysis apparatus as disclosed in co-owned, co-pending U.S. patent application Ser. No. 09/(To be amended in when received), entitled xe2x80x9cApparatus And Methods For Sample Analysisxe2x80x9d (and identified by Attorney Docket No. SYP-132), which is incorporated by reference herein. A sample analysis apparatus (or sample plug formation device) generally comprises a structure defining two channels which intersect at any angle to form a junction (or xe2x80x9cjuncturexe2x80x9d). One of the channels is a sample introduction channel having an opening for introduction of a sample. The other channel comprises a separation channel, in which may be disposed a medium capable of separating components suspected to be in the sample. The sample analysis apparatus further has means for applying a first pressure differential to the channels so that a sample flows into the junction. Subsequently, a second pressure differential moves a portion of the sample into the separation channel for separation and/or analysis. With the appropriate parameters and control of the pressure differentials, a well defined sample plug can be formed.
Thus, the present invention provides apparatus and methods for rapid, accurate, automated delivery of samples to analytical instrumentation or to chemical reagents for conducting chemical reactions. When used in conjunction with a sample analysis apparatus described above, the reaction and subsequent analysis of a sample can be a fully automated process.